2006 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Problem: Public concerns and more stringent federal regulations (including the 1996 Food Quality Protection Act) have increased the need for environmentally and toxicologically safer herbicides or other means of managing weeds. Herbicides account for more than 50% (by volume) of all pesticides used in the U.S. This percentage is likely to increase because of the rapid adoption of alternative methods for insect and fungal control and the absence of any viable alternatives to synthetic herbicides. Despite this, the availability of herbicides for minor uses, such as many horticultural crops, is diminishing. The unfavorable risk versus potential profit ratio deters agrochemical companies from investing money and manpower in the development of new compounds for this use. In some crops, such as lettuce, farmers are spending hundreds of dollars per hectare for weed management with labor- intensive methods or environmentally suspect technology such as massive use of non-biodegradable plastic mulches.
Much of the increased agricultural production of the past century is due to vastly improved pest control. At this time, the major crops in the U.S. are dependent on synthetic herbicides and the minor crops have few or no economical means of weed control, driving the cost of minor crops up. Development of environmentally benign weed management methods from natural products will reduce the risks of using synthetic compounds in major crops and provide inexpensive means of weed control in minor crops. The savings to the environment and the farmer will ultimately benefit all consumers.
Solutions: Natural products from microbes and plants offer a broad array of molecules with great diversity in their structures and biological activity. In some cases, biological activity as a phytotoxin can be utilized in weed management, either directly as a natural product-based herbicide or indirectly as an allelochemical produced by a crop or cover crop. There is considerable literature on both strategies, yet the allelochemical approach is still not adopted because of its limited capacity to control weeds and the tremendous success of synthetic herbicides. Natural product-based herbicides fit agricultural niches that the pesticide industry has little or no interest in, such as minor crops and organic gardening. There are at least three reasons why these products fit these niches. First, the risk factor is likely to be less with natural products because of their typically short half-life and often-benign toxicological profile. Second, because of these characteristics, the US Environmental Protection Agency (EPA) has a fast track registration process for natural product pest control agents (biorationals) that reduce the investment in registration. Third, the economics fit natural products. Most natural product herbicides are likely to cost more than most synthetic herbicides because of their complex structures. However, a market that already pays hundreds of dollars per hectare for weed control (versus as little as $20/ha in some agronomic crops), would be willing to pay the additional cost of such a product.
Ultimately, the most needed outcome of this work will be the development of crops that could suppress weed growth in their surroundings by producing their own herbicides. The plant biotechnology industry has expended vast resources to impart pathogen and insect resistance to crops, but we are unaware of any serious efforts to produce plants that produce their own herbicides within the private sector. Public sector laboratories throughout the world (U.S., Philippines, Korea, China, India, Japan, and Egypt) are approaching this problem with plant breeding. This strategy has not been successful in the past (see literature review). We intend to acquire the basic knowledge for both the biosynthetic enzymes and resistance mechanisms needed to produce transgenic crops that synthesize their own herbicides. Other public sector laboratories in the Far East (e.g., labs of the Japanese Ministry of Forestry, Fisheries, and Agriculture at Tskuba) are adopting similar approaches. The production of such crops is not a trivial matter, but success could reduce synthetic pesticide use in the U.S. by as much as 50%.
Much of the increased agricultural production of the past century is due to vastly improved pest control methods. At this time, the major crops in the U.S. are dependent on synthetic herbicides, and the minor crops have few or no economical means of weed control, driving the cost of minor crops up. Development of environmentally benign weed management methods from natural products will reduce the risks of using synthetic compounds in major crops, and provide inexpensive means of weed control in minor crops. The advantages to both the environment and the farmer will ultimately benefit all consumers.
This project relates strongly to two components of NP 302: Improved crop protection and enhanced crop product value. The major objective of this project is to improve crop protection from pests through the use of natural products, either as safe pesticides or as by manipulation their production in plant by genetic engineering or other means. Another objective of the project is to create value-added products from crops and/or to create new, high value crops (e.g., medicinal crops, herbal crops). The project addresses the same priorities under NP 306.
How serious is the problem? Why does is matter?
This is covered under the Problem section.
2.List by year the currently approved milestones (indicators of research progress)
Our project plan provides the milestone graphically and does not fit the 421 format. However, the milestones can be summarized as follows:
Objective 1. Discovery of natural phytotoxins.
The milestones and expected outcomes of this objective are an iterative process, involving many different phytotoxins and phytotoxin sources. At any one time, there will be projects with several compounds at different points of exploration in this research strategy.
A milestone that is a one time event is the development of a transcriptome library for phytotoxin modes of action.
Objective 2. - Identification and characterization of the biochemical and genetic pathways for the manipulation of allelopathic compounds in crops.
Milestone 1 - Elucidation of the biosynthetic pathway of sorgoleone.
Milestone 2 - Identification of the genes involved in sorgoleone biosynthesis.
Milestone 3 - Demonstration of the in vivo role of putative sorgoleone biosynthetic enzymes and metabolic engineering of the pathway.
Milestone 4 - Identification of the genes involved in the biosynthesis of lipid resorcinol in rice.
4a.List the single most significant research accomplishment during FY 2006.
The characterization of key enzymes (fatty acid desaturases) involved in the biosynthetic pathway of the allelochemical sorgoleone was accomplished. Real-time polymerase chain reaction (PCR) analysis using RNAs prepared from seven different sorghum tissues, identified three genes encoding fatty acid desaturases preferentially expressed in root hair cells. Full-length sequences were generated for all three and cloned into yeast, in which lipids were uniquely desaturated as in sorgoleone when two of the three genes were expressed. The function of the fatty acid desaturation by these cloned enzymes in other plant species was also obtained by expression in transgenic Arabidopsis. These results indicate that these genes encode the lipid desaturase genes of the sorgoleone pathway. This is part of the primary goal of our project plan that seeks to discover all of the enzymes and genes responsible for the biosynthesis of sorgoleone.
4b.List other significant research accomplishment(s), if any.
Preparation of RNA interference and overexpression vectors for manipulation of sorghum polyketide synthases involved in sorgoleone biosynthesis was completed. Isolation of promoter (5’ flanking region) sequences for both sorghum PKS genes was also accomplished. The overexpression and RNAi vectors were used to transform S. bicolor cultivar Tx430 via Agrobacterium at the University of Nebraska. Several RNAi and overexpression lines have been generated to date, and R1seed has been collected from these for analysis. A total of 10 transformant lines will be generated for each vector, and this work should be completed this year.
Molecular markers were developed to help identifying/authenticating Ephedra spp based on the sequence of the psbA-trnH spacer region.
Natural triketones from manuka oil were found to be phytotoxic and via inhibition of hydroxylphenylpyruvate dioxygenase.
Light and weeds extracts were found to have effects on sorgoleone excretion by sorghum.
The phytotoxic activity of a number of natural products was discovered. These compounds cannot be mentioned here for proprietary reasons.
4c.List significant activities that support special target populations.
Specific Cooperative Agreement between ARS and the University of Nebraska - Lincoln, the progress report is as follows:preparation of RNA interference and overexpression vectors for manipulation of sorghum polyketide synthases involved in sorgoleone biosynthesis was completed. Isolation of promoter (5’ flanking region) sequences for both sorghum PKS genes was also accomplished. The overexpression and RNAi vectors were used to transform S. bicolor cultivar Tx430 via Agrobacterium at the University of Nebraska. Several RNAi and overexpression lines have been generated to date, and R1seed has been collected from these for analysis. A total of 10 transformant lines will be generated for each vector, and this work should be completed this year.
Reimbursable Agreement between ARS and the University of Shizuoka – Shizuoka, Japan, the progress report is as follows: E. coli expression vectors containing two sorghum polyketide synthase coding sequences were transferred to the laboratory at the University of Shizuoka. In addition, an approximately 3mg crystalline sample of the physiological substrate, 16:3 fatty acyl-CoA, was provided. In turn, the laboratory expressed the two PKS proteins in E. coli and purified them, and performed a series of kinetic analyses with the two recombinant enzymes. Successful kinetic parameters were obtained for both enzymes for the 16:3 fatty acyl-CoA substrate. In addition, 16:0 fatty acyl-CoA was also tested as a control. The objectives of the research agreement were fully met, and the data will be included in a future publication.
Non-Funded Cooperative Agreement between ARS and Hawaii Agriculture Research Center, the progress report is as follows: Vulgarone (isolated from Artemisia douglasiana) and Yucca extract were tested against apple snails in tarro farms in Hawaii in collaboration with Hawaii Dept. of Agriculture. Field trials indicated very promising activity for both. Vulgarone showed activity at 75uM and yucca extract showed activity at 30 ppm. At the present time vulgarone is under consideration for further development and licensing.
Non-Funded Cooperative Agreement between ARS and The Plant Protection Research Institute, Pretoria, South Africa, the progress report is as follows: Extract from a species of Protea received from South Africa was fractionated using flash column chromatography. Pure compounds were isolated following further chromatographic work-up by preparative layer chromatography. Pure compounds were sent to South Africa for phytotoxic activity testing.
5.Describe the major accomplishments to date and their predicted or actual impact.
1) Discovery of an inexpensive method of producing podophyllotoxin from mayapple was accomplished. This discovery was patented in late 2000. We discovered that mayapple (Podophyllum peltatum) possesses a beta-glucosidase that is highly specific for aryltetralin lignans that it produces. These discoveries should provide a new high value crop for small farmers and reduce the cost of anti-cancer drugs based on podophyllotoxin. The University of Mississippi and Mississippi State University have collaborated on developing this plant as a crop because of our discovery.
2) Discovery of the molecular site of action of several natural product-based herbicides was accomplished. This research could result in the discovery and development of herbicides with new molecular target sites.
3) We discovered that plant and animals have enzymes that can metabolize protoporphyrin IX in the presence of glutathione or other thiol containing compounds. This may be a significant contributing factor for the resistance of plants to certain herbicides that cause accumulation of protoporphyrin IX. An unexpected consequence of this discovery is that this mechanism of protoporphyrin IX degradation also applies to mammals. In this case, the process we discovered may be used to develop new healthy ways of treating patients affected with a genetic disease that cause porphyrin levels to accumulate in their blood. Our discovery was reported in several articles published in popular mainstream journals.
4) The alterations (natural mutations) in the gene encoding phytoene desaturase which makes plants containing these alterations resistant to a major class of herbicides was discovered. From this knowledge, site-directed mutagenesis at the mutation site has resulted in even more resistant versions of the enzyme. These findings have resulted in a patent of the gene for two potential uses: .
1)an improved selectable transformation marker and.
2)production of crops resistant to certain phytoene desaturase herbicides. One major company is in the process of licensing this technology as a selectable marker.
5) The genes for biosynthesis of the allelochemical sorgoleone have been identified. This accomplishment is fundamental to carrying out the molecular biology and biochemistry that will be needed to manipulate the synthesis of this potent allelochemical in crops for better weed management. Some of these genes have other potential uses. Two patents on these genes for these uses are in progress.
6) Transcription profiling of fungicide mode of action with whole genome microarrsays showed that ergosterol synthesis inhibitors have characteristic transcription profiles for the 24 genes in the ergosterol synthesis pathway. Similarly, a methionine synthesis inhibitor affected genes of the methionine synthesis pathway.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
The one patented technology from the first five years of this project has been patented, but not licensed.
The phytoene desaturase gene being patented with SePRO is now at the U.S. patent office. One company is considering licensing it.
A gene for improving the nutriceutical properties of small fruit is being patented.
A gene for altering plant lipids in a novel way is being patented.
The time line on getting this technology to the farmer is 6-10 years, mainly due to regulatory constraints.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Symposium presentation entitled, “Herbicide Effects on Plant Disease”, at 25th Brazilian Weed Science Congress in Brasilia, Brazil, in June, 2006.
Seminar entitled, “Pest Management Phytochemicals: Strategies for Discovery and
Utilization” at the Center for Plant Cell Biology at the University of California, Riverside in June, 2006.
Symposium paper entitled, “ Discovery and Development of Natural Products for Pest Management” at the Phytochemical Society of North America annual meeting in Oxford, MS, in July, 2006.
Symposium paper entitled, “Transgenes for Weed Management” at the 4th Annual Meeting of Special Committee on Agrochemical Bioscience of the Pesticide Science Society of Japan, a Satellite Symposium of the IUPAC ICPC 2006 Conference held in Tokyo, Japan, in August, 2006.
Symposium paper entitled, “Allelochemicals for Weed Management: Molecular Biology Approaches” at the IUPAC 11th International Congress of Chemistry in Crop Protection held in Kobe, Japan, in August, 2006.
Dayan, F.E. 2006. Factors modulating the levels of the allelochemical sorgoleone in sorghum bicolor. Planta. 224:339-346.
Baerson, S.R., Dayan, F.E., Rimando, A.M., Pan, Z., Cook, D., Nanyakkara, N., Duke, S.O. A functional genomics approach for the identification of genes involved in the biosynthesis of the allelochemical sorgoleone. Rimando, A.M., Duke, S.O., editors. American Chemical Society, Washington, DC. Natural Products for Pest Management. 2006. p. 265-277.
Inderjit, Weston, L.A., Duke, S.O. 2006. Challenges, achievements and opportunities in allelopathy research. Journal of Plant Interactions. 1(2): 69-81.
Kagan, I., Michel, A., Scheffler, B.E., Prause, A., Baerson, S.R., Duke, S.O. Global gene expression approaches to mode-of-action studies with natural product-based pesticides. Rimando, A.M., Duke, S.O., editors. American Chemical Society, Washington, DC. Natural Products for Pest Management. 2006. pp. 255-264.